The limited capacity of the injured myocardium to reactivate cardiomyocyte (CM) proliferation is a major barrier to myocardial regeneration. Stem cell derived CMs are considered as an alternative cell source to replace damaged CMs. Various types of stem cells are under investigation of their potential for CM differentiation. Skeletal muscle derived stem cells (MDSCs) are a somatic stem cell population obtained from skeletal muscle specimens. MDSCs have been shown to differentiate along multiple lineages including skeletal muscle, bone, tendon, nerve, endothelial, hematopoietic, and smooth muscle cells. MDSCs are easily expanded in vitro and can be transplanted as autologous grafts. Previous studies, including our own work, have shown that MDSCs have potential for cardiac repair and can differentiate into CMs. We have recently succeeded in the induction of functioning CM from cultured rat MDSCs using the strategy of combined MDSC-aggregate sphere formation and MDSC-sphere-3-dimensional cardiac gel bioreactor (MDSC-sphere-3DGB). MDSC-sphere-3DGB is scalable and the MDSC-derived CMs are easily extracted by enzymatic digestion of the construct. MDSC-sphere-3DGB also allows the direct evaluation of MDSC-derived CM contractile properties as an engineered cardiac tissue. Thus, the aim of the proposal is to optimize a tissue culture method to obtain functioning CMs differentiated from human MDSCs. Specific Aim: Define the optimal condition of human MDSC-sphere-3DGB for functioning CM induction. We will test 4 hypotheses, 1). Human MDSCs differentiate into functioning CMs using a MDSC-sphere-3DGB system;2). Additive retinoic acid treatment in MDSC-sphere-3DGB further increases functioning CM differentiation;3). Cyclic mechanical stretch stimulation increases human MDSC-derived CM proliferation and contractile properties similar to the immature fetal type CMs;4). Cyclic electric stimulation increases human MDSC-derived CM maturation and improves tolerance to the hypoxic conditions. Significance: The goal of the present proposal is to establish a paradigm using a MDSC-sphere-3DGB system to define the differentiation of the functioning human MDSC-derived CMs. These studies support our long term goal of implementing autologous cellular cardiomyoplasty to repair congenital and acquired heart diseases. PUBLIC HEALTH RELEVANCE: We have recently succeeded in generating workable heart muscle cell from rat skeletal muscle derived stem cells using a 3-dimensional tissue culture system (bioreactor). The skeletal muscle derived stem cell is relatively easily obtained from a skeletal muscle biopsy of heart disease patients and the induced heart muscle cells from the skeletal muscle stem cells can be transplanted to the same patients to cure the damaged heart. This proposal is to investigate the optimal conditions of HUMAN skeletal muscle derived stem cell-derived heart muscle cells to cure congenital and acquired heart diseases.